1. Field of the Invention
The present invention relates to a method and composition for determination of glycerol, and particularly to a method and composition for determination of glycerol by using glycerol dehydrogenase, characterized in that the glycerol dehydrogenase-catalyzed reaction for the determination is promoted by dihydroxyacetone kinase or triokinase. The invention is specially useful for the determination of glycerol and triglyceride be contained within biological fluids.
2. Description of the Prior Art
The determination of glycerol and triglyceride contained within blood is very important for diagnosis of hyperlipemia. In particular, hypercontent of triglyceride in blood is characteristic of arteriosclerosis, coronary insufficiency, myocardial infarction, etc. For early diagnoses or treatments of these diseases, a rapid and accurate method is desired for determination of triglyceride content in blood.
A method for determination of triglyceride using lipase and glycerol dehydrogenase is characterized by the following reaction sequence. ##STR1## NAD(P).sup.+ represents nicotinamide adenine dinucleotide (phosphate), NAD(P)H represents reduced NAD(P).sup.+, and GDH represents glycerol dehydrogenase.
First, triglyceride is hydrolyzed into glycerol and fatty acids by the action of lipase, then the glycerol is oxidized into dihydroxyacetone (or D-glyceraldehyde) in the presence of NAD(P).sup.+ by the action of glycerol dehydrogenase. The formed NAD(P)H is measured by spectrophotometrical or fluorometrical assay, thereby determining glycerol and consequently triglyceride.
The equilibrium of the above GDH-catalyzed reaction is shifted in the reverse direction. Thus, the reaction does not sufficiently proceed in the forward direction. In addition, it takes a long time to attain equilibrium. Consequently, the measurement is unsatisfactory in precision or sensitivity, and the range of measurable glycerol or triglyceride concen- trations.
In order to overcome the above difficulty, it was necessary to carry out the reaction in as high pH as from 10 to 11, or to add excess NAD(P).sup.+ to the reaction mixture.
A possible preferable approach to the promotion of the above GDH-catalyzed reaction is to eliminate the product dihydroxyacetone or D-glyceraldehyde from the assay system. For example, the method of converting the dihydroxyacetone into the corresponding hydrazone by adding hydrazine was attempted to promote the GDH-catalyzed reaction [Rinshobyori (Clinical Pathology), 24, 855 (1976)]. However, this method is unsatisfactory, since the enzyme is inactivated by hydrazine during the reaction.